Development of the vertebrate musculoskeletal system requires the coordinated morphogenesis of muscle, muscle connective tissue, tendon, and skeleton. In the limb, muscle derives from migratory precursors originating from the somites, while the muscle connective tissue, tendons, and skeletal elements derive from the mesoderm of the emerging lateral plate-derived limb bud. As muscle precursors migrate into the limb, they differentiate into myofibers, become correctly patterned into distinct muscles, and are assembled with muscle connective tissue, tendons, and bones into a functional musculoskeleton. How muscles and tendons are patterned and assembled with muscle connective tissue is largely unknown, defective in multiple human genetic syndromes, and the subject of this proposal. The close developmental association of muscle and tendon with connective tissue suggests that interactions between these tissues may be critical for their development. However, study of muscle connective tissue has been hindered by the lack of molecular markers and genetic reagents to label connective tissue fibroblasts. We identified that the transcription factor Tcf4 (Tcf7L2) is strongly expressed in adult connective tissue fibroblasts and in their precursors in the embryo. During the previous grant period, we engineered Tcf4GFPCre and Tcf4CreERT2 mice, the first reagents to allow for genetic manipulation of these fibroblasts. Our preliminary studies of Tcf4 function suggest that the connective tissue regulates muscle and tendon morphogenesis in the developing limb. In addition, we and our collaborators have found that Lmx1b, Tbx3, Tbx4, and Tbx5 are key transcription factors that establish the dorsal/ventral, anterior/posterior, and fore/hindlimb asymmetries of the limb and determine the pattern of limb muscles and tendons. Preliminary studies suggest that these transcription factors regulate muscle and tendon morphogenesis non-cell autonomously via their function in muscle connective tissue. Here we propose to test the hypothesis that connective tissue, via Tcf4, Lmx1b, Tbx3, Tbx4, and Tbx5 function, determines the pattern of limb muscles and tendons. These experiments will elucidate the cell-cell and molecular interactions necessary to coordinate and assemble an integrated and functional musculoskeletal system. Furthermore, mutations in Lmx1b, Tbx3, Tbx4, and Tbx5 have been identified as the genetic causes of human Nail-Patella, Ulnar-Mammary, Small Patella, and Holt-Oram syndromes, characterized by severe abnormalities in limb muscle, tendon, and skeleton. How these genetic mutations cause these musculoskeletal defects is unknown. Our analysis of mouse mutants of these genes, which phenocopy the human syndromes, will elucidate the cellular and molecular defects that lead to these limb musculoskeletal abnormalities.